There is a need for novel simple assay formats for sensitive DNA sequence analyses as well as for detection of RNA and proteins, suitable for e.g. a clinical setting, bed-side applications, military purposes, forensic analyses, food and pharmaceutical product analyses, as well as analyses of microorganisms in the environment.
Below, the state of the art of DNA sequence analysis is briefly recapitulated.
Information about the presence and/or nature of DNA sequences is of importance in many areas such as e.g. in clinical diagnostics, to guide therapy and make accurate diagnosis of disease and in analysis of microorganisms present in the environment. Recently, immunoassay techniques have emerged that use DNA as reporter molecules to indicate detection of specific protein analytes, making DNA analysis techniques useful for all biomolecules of interest for diagnosis (IMMUNO-PCR [Sano et al.], IMMUNO-RCA [Schweitzer et al.], Bio-barcodes [Nam et al.], PLA [Fredriksson et al.]). DNA sequence detection schemes often rely on a hybridization reaction between a target-DNA molecule and a probe molecule, designed to match the target. Large-scale DNA hybridization analyses are efficiently performed using high-density microarrays of synthetic oligonucleotide probes and optical readout using e.g. fluorescence labelling and fluorescence detection, quantum dot labelling and metal colloid labels taking advantage of surface plasmon scattering phenomena [Bally et al.]. Microarray analysis is relatively labour-some and time-consuming, and limited in sensitivity and specificity, and is therefore not very suitable for diagnostics. It is an advantage if hybridizations can be monitored in homogenous readout formats that do not require separation of unbound labelled probes from the matching probe-target complexes. One commonly used technique to achieve this is based on detection of the change in fluorescence depolarization when a probe binds its target. Other readout formats for hybridization reactions, more suitable for diagnostics, have been explored. These involve e.g. electrochemical detection schemes [Kerman et al.], where monitoring of the hybridization reaction is based on the electrochemical response when the probes, labelled with e.g. organic dyes, metal complexes and metal nanoparticles, bind their targets. There also exist gravimetric detection schemes such as micro-cantilever resonance-based DNA detection with nanoparticle probes [Su et al.]. To achieve high detection sensitivity—in the extreme case single molecule detection—it is typically necessary to amplify the probe-target complex. One of the most commonly used methods for this is the polymerase chain reaction (PCR) [Saiki et al.]. Although the PCR scheme in principle provides unlimited sensitivity and quantitative dynamic range, the technique is considered too complicated for the diagnostic setting.
An alternative approach for enzymatic detection and amplification for detecting sets of gene sequences with high specificity and selectivity involves the use of circularizing oligonucleotide probes (padlock probes) [Nilsson et al. 1994, Landegren et al.] for recognition of the target-DNA in combination with enzymatic signal amplification by the RCA mechanism [Fire and Xu, Liu et al.]. The 5′ and 3′ ends of the linear padlock probe are designed to base-pair next to each other on the target strand. Thereafter, if properly hybridised, the ends can be enzymatically joined by a DNA ligase, thereby creating a circularly closed probe-target complex (reacted probe) for each recognised target. Circularized probes can then be amplified by a DNA polymerase using the RCA mechanism, which generates a DNA strand consisting of a large number of tandem copies of the complement to the circularized probe, collapsing into a random-coil single-stranded DNA macromolecule in solution [Blab et al.]. The RCA process can also be executed by an RNA polymerase creating a corresponding long concatemer RNA strand [Daubendiek and Kool]. Jarvius et al. demonstrated this scheme for DNA single molecule detection. The RCA-products were detected by using fluorescence molecule-tagged probes, designed to hybridise to the repeated sequence of the RCA-product, resulting in a confined cluster of fluorophores [Jarvius et al.]. These clusters were in turn detected and quantified by pumping the sample through a microfluidic device mounted in a standard confocal fluorescence microscope operating in line-scan mode, thereby allowing for digital quantification [ibid]. Furthermore, various circularized probe-target complexes, each corresponding to a unique target sequence, could be formed and amplified simultaneously [ibid]. Hybridization of fluorescence probes with different colours provided the opportunity to perform multiplexed target analysis [ibid]. Although fluorescence detection of the RCA-products, the current state-of-the-art method for DNA sequence detection, has several advantages such as high selectivity and sensitivity, the equipment needed for this is expensive and rather difficult to miniaturize.
Biomolecule detection by measuring changes in the Brownian relaxation frequency of magnetic nanoparticles in aqueous solution was originally proposed by Connolly and St Pierre. In their detection scheme, the surfaces of magnetic nanoparticles are biofunctionalized with probe molecules, e.g. single-stranded oligonucleotide molecules. When single-stranded DNA molecules having a sequence that matches the probe oligonucleotides are added, hybridization reactions occur on the surface of the magnetic nanoparticles, giving rise to an increased hydrodynamic diameter and consequently, a decreased Brownian relaxation frequency. This detection principle has been demonstrated in the case of antigen-antibody reaction by Astalan et al. The complex magnetization spectra were recorded using induction coils and a lock-in amplifier technique. Another example of a magnetic nanoparticle based scheme without fluorescence read-out is the high sensitivity InSb Hall effect biosensor for DNA detection using functionalised magnetic nanoparticles [Togawa et al.] and the giant magnetoresistive biosensor device for magnetic DNA detection [Tamanaha et al.]. Common for the above described magnetic detection schemes are that a rather large number of probe-target hybridization events on the surface of the magnetic nanoparticles are required to produce a reliable output signal.